Biological interaction/activities, such as protein:protein interactions, antigen:antibody interactions, protein:nucleic interactions, protein:ligand interactions and nucleic acid:nucleic acid interactions are involved in a wide variety of processes occurring in living cells. For example, agonism and antagonism of receptors by specific ligands, antibody-antigen interactions, including drugs, hormones, second messenger molecules, etc. may effect a variety of biological processes such as gene expression, cellular differentiation and growth, enzyme activity, metabolite flow and metabolite partitioning between cellular compartments, amongst others. DNA:protein and RNA:protein interactions are well known for their effects in regulating gene expression in both prokaryotic and eukaryotic cells, in addition to being critical for DNA replication and in the case of certain viruses, RNA replication. In cases where the propagation of cells is deleterious such as the replication of a pathogen or of a cancer cell, agents which target biological interaction/activities or functional structures, are suitable candidates for therapy. For example, agents that block the function of membrane channels or disrupt cytoplasmic membranes by other means, are attractive targets for anti-microbial therapies against pathogens. Further, agents that interact with antigen-specific or non-specific functions of the immune system may provide immunological modulators or vaccines for allergy, autoimmunity, infectious disease, fertility and invenomation. For example, agents that have the antigenicity of microbial antigens, tumour antigens, allergens or autoantigens may be used for vaccines or immunotherapy.
Undesirable or inappropriate gene expression and/or cellular differentiation, cellular growth and metabolism may be also be attributable, at least in many cases, to biological interaction/activities involving the binding and/or activity of proteinaceous molecules, such as transcription factors, peptide hormones, receptor molecules and enzymes, amongst others. In these cases, therapies can be envisaged which block such inappropriate interactions and/or which block the formation of inappropriate cellular structures.
Production of Peptides by Recombinant DNA Techniques
Peptides that can mediate or interfere with a diverse range of biological functions include natural peptides and peptides synthesised to represent a portion or a modified portion of a molecular known to mediate a target function. One source of such peptides are random peptides libraries constructed with random (or semi-random) oligonucleotides ligated into cloning sites of a plasmid or phage vectors.
Vectors containing DNA encoding different peptides are transfected or transformed into bacteria or other hosts and cloned by standard plaque or colony purification procedures. Clones producing peptides with a desired activity can be isolated by a variety of screening or selection procedures which are fundamentally the same as the screening procedures used to detect polypeptides encoded by cDNA or cDNA fragments. These include the production of peptides as fusions with the coat proteins of bacteriophage or fusions with bacterial surface proteins so the peptides can be used as tags for affinity purification procedures; the production of peptides from hosts infected with phage or transformed with plasmids to produce arrays of colonies or plaques which can be screened for ligand-binding activity or biological activity such as inhibiting the growth target bacteria or inducing the activation of genes in target bacteria; and in positive selection strategies such as the two hybrid cloning systems, where the peptide produced in the host microorganism binds to target proteins to form complexes which activate the expression of the reporter genes cloned into the same host. One of the significant advantages of phage display technology is that it enables the construction of libraries with very large complexities—ie. 1010 to 1011 individual clones.
Likewise, in ‘reverse two hybrid’ or ‘split two hybrid’ systems, libraries of appropriately expressed peptides can be screened for blockers of particular protein/protein interactions, which in turn reduces the expression of counter selectable reporter genes encoding toxic products.
Modifications of Peptides for Utility and Optimisation
Once the active peptide or a ligand binding peptide has been identified they can be modified by a variety of procedures to optimise their utility. Modification may include: alterations in the amino acid residues which engage the target to improve their binding specificity and affinity; modifications which affect the display of the peptide including the valency of binding and constraint of particular conformations; and modifications to attach further functional moieties such as markers, toxins and co-activators.
Synthetic peptides can include residues other than the 20 amino acids found in nature and/or can be cyclised by means such as oxidation of flanking cysteine residues. In the case of peptides mimicking antibody epitopes, carriers containing the T-cell epitopes required to induce high affinity immune responses can be added by genetic techniques.
Example of Peptides that Modulate Biological Systems
Peptides can be applied as therapeutics or lead molecules for designing therapeutics for disease including infection, cancer and metabolic disorders as well as agents for vaccines and immunotherapy, transplantation and diagnostics. The potential usefulness of such peptides has been demonstrated by the following examples:
Peptide Antimicrobial Agents
The antimicrobial effect demonstrated by natural peptides produced by frogs and insects and artificially synthesised cationic peptides. A large variety of antibiotics are peptides or polypeptides. The granules of mammalian neutrophils produce families of antimicrobial polypeptides including azurocidin, cathepsin G and Cationic Antimicrobial Peptides (CAP57 and CAP37). In addition, neutrophils produce at least two families of antimicrobial peptides, the defensins and the bactenecins. Moreover, many natural antibiotics and antifungal drugs are composed of peptides. For example, the magainin family of antimicrobial α-helical peptides isolated from the skin of the African clawed toad, Xenopus Laevis form lethal pores in the cell membranes of certain microorganisms. Similarly, certain α-helical peptides derived from a variety of insect genera have antimicrobial activity. Recently, several rational design approaches have been used to isolate novel peptide antibiotics. For example, Tiozzo et al., used a “sequence template” approach in which candidate peptide sequences were designed from alignments of natural antimicrobial peptides [1]. The identification of virulence determinants in several pathogens presents other attractive targets for antimicrobial therapy. For example, Balaban and colleagues (2) have recently identified an autoinducer of virulence in Staphylococcus aureus that controls the production of bacterial toxins involved in pathogenesis. The toxin genes are induced by a regulatory RNA molecule, RNAIII that is induced by a threshold concentration of an endogenous protein RNAIII Activating Protein (RAP) [2]. Peptide inhibitors of RAP might be expected to act as virulence determinants. Indeed, a natural peptide inhibitor of RAP called RIP (RNAIII inhibiting peptide) is produced by a non-pathogenic strain of Staphylococcus aureus and appears to inhibit the RNAIII gene and to cause reduced virulence [2].
Peptide Modulators of Growth Regulation
The ability of peptides to affect key modulators of growth regulation has been demonstrated by Brent and colleagues who used two hybrid screening to identified constrained peptide ‘aptamers’ from combinatorial libraries which bind tightly to and inhibit the function of cyclin dependent kinase 2. This demonstrates the potential for treatment of neoplasms (3).
Peptides can exhibit exquisite specificity. For example, peptide aptamers have been identified which can discriminate between two closely related allelic varients of the Ras protein (4). Moreover, a peptide aptamer against human cyclin dependent kinase 2 inhibits kinase activity exclusively on certain particular substrates.
Peptide specificity has also been demonstrated in vivo. In a recent report, expression of aptamers that recognised cyclin dependent kinases in transgenic flies was shown to cause developmental abnormalities in a dominant negative fashion (5). Importantly, the specificity of the two aptamers for particular Cdks (as determined by yeast two hybrid assays) was retained in the Drosophila in vivo assay. Moreover co-expression of the specific aptamer target Cdk suppressed the developmental phenotype observed (5). This report of successful targeted inhibition of an enzyme in vivo with aptamers, firmly establishes as practicable the principle for developing new therapeutic strategies based on interfering peptides.
Peptide-Based Inhibition: An Emerging Therapeutic Strategy
Much attention has recently focussed on peptides as potential therapeutic agents because they can be highly specific and readily synthesised. Phase display technologies are beginning to prove useful for providing peptide leads in drug discovery programs. Efficient delivery of peptide from outside the cell to the nucleus of eukaryotic cells can now be achieved by attaching sequences such as the targeting motif “penetratin” which is derived from the Drosophila Antennapaedia protein. More recently a family of such targeting peptides has been identified (6). For example, conjugation of peptide sequences to the VP22 protein has been shown to allow efficient export of the fusion protein to the nuclei of cells adjacent to primary transfectants (7). Several recent developments make it feasible to physically select conformationally constrained peptide domains in order to identify peptides that bind with very high affinity in vivo, favouring high potency. Mimetic peptides have been reported to inhibit protein interactions and/or enzyme function. Examples include a nonapeptide derived from the ribonucleotide reductase of herpes simplex virus that was linked to an enterotoxin subunit for delivery into cells via its receptor. The peptide conjugate was found to inhibit herpes simplex type 1 replication in quiescent Vero cells [8]. Using detailed knowledge of the PCNA-interaction domain of p21WAF1 derived from two hybrid screens, a peptide has been designed which effectively block the interaction. This 20-mer bound with sufficient affinity to block SV40 replication. A 20-mer peptide sequence derived from p16 has been found to interact with Cdk4 and Cdk6 and inhibited pRB phosphorylation and cell cycle progression [9]. The authors coupled the specific inhibitor peptide to the 16 residue penetration peptide for efficient nuclear delivery. Peptides have even been shown to function as inhibitors in animal models. For example, a tetrapeptide mimicking the substrate of farnesyl protein transferase has also been shown to block the growth of Ras-dependent tumours in nude mice.
Peptide Mimotopes
Peptides functionally resembling the epitopes (mimotopes) bound by antibodies have been isolated and used as experimental vaccine to induce antibodies which protect against infection as shown for hepatitis B, respiratory syncytial virus, Japanese encephalitis and Streptococcus pneumonia. High affinity antibodies typically bind complex structures formed by the tertiary conformation of an antigen. The peptide mimotopes essentially convert a conformational epitope made from a complete protein into a small peptide. It has advantages when only certain epitopes are desired, e.g. to prevent immunopathology in RSV infection; or in the production of recombinant epitopes where the complete polypeptide may be difficult to fold; or where the entire antigen has undesirable biological properties (Staphylococcal toxins in toxic shock syndrome). In the case of carbohydrate antigens, polypeptides that contain the mimotope can be constructed to convert a T-cell independent antigen into a T-dependent antigen for the production of high affinity antibodies and immunogenicity in young animals including humans. Unlike the carbohydrates, peptide mimotopes can be produced as DNA vaccines.
The possibility of using mimotopes as antigens for cancer immunotherapy has been demonstrated for an adenocarcinoma antigen.
Mimotopes can be used as antigens to diagnose infectious disease by detecting antibody. The possibility has been demonstrated with hepatitis C infection.
Mimotopes representing the antigens recognised by autoantibodies against β-islet tissue in diabetes have been demonstrated and it has been proposed that these could be used to monitor the development of disease (10). Similarly mimotopes have been found for pollen allergens which could be used in the diagnosis of allergic disease. In both these cases it is also possible that the mimotopes could be used for therapy by modulating the immune response or in prophylaxis.
Mimotopes representing transplantation antigens have been demonstrated and thus may be used as tolerogens or blockers to prevent transplantation rejection.
Ligand Interactions or Hormone Receptor Interactions
Peptide mimetics have been used as ligands to affinity purify biologically useful molecules as shown for the purification of the blood clotting protein, von Willebrand factor.
The modification of enzyme activity with peptides mimicking substrates has also been demonstrated. Peptide mimetics can be used as hormones as shown for erythropoietin and can be modified to increase biological activity.
Recombinant Methods for Producing Biological Active Peptides
The use of fragments from specific genes or cDNA to produce peptides containing a biological activity of the polypeptide encoded by the gene or an inhibitor of the activity can sometimes be successful. In other instances the activity can be dependent on the conformation of complete polypeptide and cannot be obtained by these techniques. In many cases the use of random peptide libraries in phage or plasmids to produce a peptide which mimics the biological activity has been successful. This involves the screening of large numbers of clones producing an essentially random array of peptides for a peptide of the desired activity.
The activity is sometimes mediated by a peptide which shows an amino acid sequence homology which could explain its biological activity while in many cases the peptide acts as a mimetic for the conformation of the polypeptide or its ligand and has no sequence homology. Indeed the peptide may be a mimetic of a chemically different molecule such as a carbohydrate. It is also possible to use the combinatorial library approach to screen for inhibitors or mediators of complex functions where there is no information on the molecular interactions required.
The ability to isolate active peptides from random fragment libraries can however be highly variable and problems with low affinity interactions have been reported, particularly for peptides required to represent complex conformations such as discontinuous epitopes bound by many antibodies. There is unpredictability in that, libraries that are a rich source of peptides for one ligand may not contain peptides for others. While the ability to obtain desired peptides should be increased with libraries containing larger random peptides and more random peptides there are practical difficulties in conducting high throughput screening or affinity purification particularly since it has been shown that high-density affinity purification is inefficient. There is also uncertainty about the degree to which peptides isolated from the random peptide libraries will retain their binding or biological activity when produced as part of different delivery strategies such as fusions with different polypeptides. There is thus an opportunity to supplement or improve the existing technology with new strategies.
Biodiverse Peptide Domain Libraries from defined Genomic Sources
Peptides present potential therapeutic and prophylactic agents for many human and animal diseases, biochemical disorders and adverse drug effects, because they can interact with other molecules with high specificity and affinity. However, a major problem to be overcome in the field of peptide therapeutics and prophylactics is the identification of specific amino acid sequences having a desired antagonist or agonist activity against a particular biological activity in a particular cellular environment. Such candidate peptide drugs may be particularly difficult to identify from truly random peptide libraries that lack any enrichment for sequences encoding molecular shapes suitable for binding biological structures. In contrast, nature has already assembled a rich source of such domains within the myriad of peptides, polypeptides and proteins encoded by the diverse range of genomes that make up the biosphere.
A wide range of different methods have been put forward to facilitate the screening of biological libraries (such as cDNA libraries) in an expedient manner to identify suitable protein or polypeptide molecules. Libraries of thousands and in some cases even millions of polypeptides or peptides have been prepared by gene expression systems and displayed on chemical supports or in biological systems suitable for testing biological activity. Generally such libraries are made from either individual genomes of organisms believed to be rich sources of new drugs (such as ‘extremophile’ bacterial species) or from a mixture of uncharacterised genomes isolated directly from the environment.
While the screening of biodiverse libraries has proven valuable, such libraries tend to be biased towards the frequency with which a particular organism is found in the native environment and may not necessarily represent the true population of the biodiversity found in a particular biological sample. Moreover, such screens are normally intended to isolate genes encoding enzymes, hence attempts are often made to bias such libraries to contain larger inserts which could be expected to encode biologically active enzymes.
In U.S. Pat. No. 5,763,239 in the name of Short et al., a procedure is described for normalising genomic DNA libraries from an environmental sample, in an attempt to address this problem of bias. Because the libraries mentioned in that patent are generated from environmental samples for which little would be known about the genomic constitution of the library the procedure employs complicated normalisation methods to normalise the genomic constitution of the libraries. While that procedure permits some normalisation of the genomes in an environmental sample, the methods that is describes are complicated, there is a risk that rare genomic DNA's will be lost when the methods are applied and/or that new biases will be introduced by the procedure.
In addition to the above, current screening methods often rely on the isolation of genomic nucleic acid sequences using PCR amplification procedures for which little may be known about the genomic sequences. In such cases biases can be introduced through such factors as the presence of disproportional representation of repeated sequences in certain genomes. Furthermore, because no information is known about the genomic constitution of the environmental sample, only limited bioinformatic data can be derived from a screen of the library. This problem is addressed to some extent in U.S. Pat. No. 5,763,239, which seeks to increase the probability that a genomic sequence of low copy number in an environmental sample will have a chance of being represented in a library.
There are, however, currently no available methods for screening normalized biodiverse peptide domain libraries in vivo wherein the entire composition and complexity of the library can be accurately estimated and wherein the screening process provides such comprehensive bioinformatic data useful for rational drug design. Moreover, no methods have been described with are specifically designed for the construction of natural genomic sequence libraries that have been optimised for the expression of domains per se, rather than entire polypeptides. Accordingly, there is a need to develop technologies that provides for the large-scale screening of peptide libraries which are enriched for sequences encoding bioactive domains useful in the determination of useful peptide therapeutics, the basis of which is not necessarily related to the natural role of particular peptide domains.